The overall goal of the research proposed is to develop a detailed understanding about the biology of novel small RNA species in E. coli and yeast. Of special interest are the metabolically stable 4.5S RNA of E. coli and the small nuclear RNAs (snRNA) of Saccharomyces cerevisiae. the major focus for each RNA is discovering and defining its biological function. Early evidence points to a role in translation for the 4.5S RNA species and one of the snRNA species (snR128) shows promise for a role in mRNA splicing. The 4.5S RNA has a particularly unique structure and this will also receive attention. A universal plan has been developed for identifying small RNA function that is based on both genetic and biochemical strategies. The approaches include: 1) evaluating the physiological effects of individual small RNA loss, 2) functional mapping of the RNAs by mutagenesis, 3) characterization of unlinked RNA-minus suppressor mutants, to identify interacting molecules, 4) preparation of antibodies to small RNA binding proteins and use of these probes to interfere with function and characterize native RNA-protein complexes. The specific aims for the E. coli 4.5S RNA are: 1) to identify specific 4.5S RNA binding proteins, 2) to characterize the 4.5S RNA requirement in translation, 3) to develop a functional map of the 4.5S RNA and 4) to characterize the solution structure of the 4.5S RNA molecule. The structural studies will include scanning microcalorimetry and proton NMR. For the snRNAs the aims are: 1) to identify the role of the essential snR128 (consisting of 128 nucleotides), 2) to identify proteins that bind to the snRNAs of the spliceosome and characterize the role of spliceosome snRNPs in mRNA maturation and 3) to describe new snRNA species and their genes. Early emphasis with the snR128 species will be on a) assessing the importance of sequences complementary to two intron domains required for splicing, b) characterizing three extragenic SNR128 suppressors that appear to correspond to genes known to be required for mRNA splicing (i.e. the rna loci) and c) characterizing snR128 binding proteins. Results from the proposed studies should reveal important insights into the biological roles of small RNAs in other cells and establish general approaches for characterizing these vital species in higher organisms, including humans.